Varicella-zoster virus (VZV) is the cause of chickenpox and herpes zoster. VZV infection is a recurrent problem in both transplant and cancer patients. Herpes zoster and post herpetic neuralgia are also major medical problems in the elderly population. The VZV genome encodes about 70 open reading frames. The predominant VZV glycoprotein complex, in contrast to other alphaherpesviruses, is gE:gI. The long-term goal of this project is an in depth understanding of the role of VZV gE glycoprotein, an essential gene product which has many attributes of a cell surface receptor. For example, gE has endocytosis signals and both serine/threonine and tyrosine phosphorylation motifs in its cytoplasmic tail. The endocytosis of gE is clathrin mediated; subsequent gE trafficking is dependent on a casein kinase II phosphorylation sequence and the association of PACS-1. Analyses with a recombinant biologically active VZV ORF47 protein serine kinase led to the discovery that the viral kinase has a similar phosphorylation consensus motif to that of casein kinase II. Therefore, the hypothesis states that the ORF47 protein kinase and casein kinase II will act as team players to modify the acidic gE consensus site and in the process determine the gE trafficking pathways. The Specific Aims include the following: 1) Characterize the phosphorylation of gE by ORF47 kinase and document differences from casein kinase II phosphorylation, 2) Investigate the trafficking of gE in the presence and absence of ORF47 kinase, 3) Characterize the tyrosine phosphorylation sites on gE and the cellular site of tyrosine phosphorylation, and 4) Investigate the interaction of gI with gE during the phosphorylation and trafficking events, and the interaction of the gE:gl complex with PACS-l. Phosphorylation analyses will include both in vitro kinase assays and 2-D phosphopeptide gels. The trafficking experiments will include both transfection studies with wild type and mutant gE and gI glycoproteins, as well as infection studies with recombinant viruses containing mutated gE and ORF47 proteins. Endocytosis and trafficking data with labeled viral and cellular proteins will be analyzed by confocal microscopy followed by digital image analysis to obtain a pixel quantification. Preliminary data with wild type and mutant VZV suggested that gE trafficking was redirected in cells infected with an ORF47 mutant virus, namely, gE did not travel to the trans Golgi. Thus, this latter experiment provided strong evidence of an inter-relationship between virus-specific phosphorylation and directed trafficking of the gE glycoprotein. In turn, the cumulative data suggest that VZV gE trafficking occurs in two phosphorylation dependent pathways: one pathway not directed to the TGN may facilitate cell spread while the other pathway directed to the TGN may facilitate glycoprotein incorporation into virions. These proposed VZV studies will provide further insight into the multiple functions of gE and the gE:gI complex.